The present invention relates to proteins, especially proteins that are capable of degrading starch.
In particular, the present invention relates to the use of proteins that are capable of retarding the detrimental retrogradation of starch.
Detrimental retrogradation processes, such as staling, typically occur after the heating and cooling of starch media, in particular aqueous starch suspensions, and are due to transformation of gelatinised starch to an increasingly ordered state.
More in particular, the present invention relates to the use of proteins that are capable of retarding the detrimental retrogradation of amylopectin.
More in particular, the present invention relates to the use of proteins to prepare baked bread products, as well as to the baked bread products themselves.
More in particular, the present invention relates to retardation of staling in baked farinaceous bread products.
More specifically the present invention relates to a process for making a baked farinaceous bread product having retarded or reduced staling, comprising adding a non-maltogenic exoamylase to the bread dough.
The present invention also relates to an improver composition for dough and baked farinaceous bread products comprising a non-maltogenic exoamylase.
Starch comprises amylopectin and amylose. Amylopectin is a highly branched carbohydrate polymer with short xcex1-(1xe2x86x924)-D-glucan chains which are joined together at branch points through xcex1-(1xe2x86x926) linkages forming a branched and bushlike structure. On average, there is one branch point for every 20-25 xcex1-(1xe2x86x924) linked glucose residues. In contrast, amylose is a linear structure mainly consisting of unbranched xcex1-(1xe2x86x924)-D-glucan units. Typically, starches contain about 75% amylopectin molecules and about 25% amylose molecules.
More specifically, linear malto-oligosaccharides are composed of 2-10 units of xcex1-D-glucopyranose linked by an xcex1-(1xe2x86x924) bond. Due to their properties such as low sweetness, high waterholding capacity, and prevention of sucrose crystallisation [1] these compounds have potential applications in the food industry. The preparation of malto-oligosaccharides with a degree of polymerisation (DP) above 3 (i.e. DP greater than 3) in larger amounts is however tedious and expensive.
As background information, DP1=glucose, DP2=maltose, DP3=maltotriose, DP4=maltotetraose, DP5=maltopentaose, DP6=maltohexaose, DP7=maltoheptaose, DP8=maltooctaose, DP9=maltononaose, and DP10=maltodecaose.
The discovery of microbial enzymes, which produce malto-oligosaccharides of a specific length could allow the production of larger amounts of these oligosaccharides [2].
Amylases are starch-degrading enzymes, classified as hydrolases, which cleave xcex1-D-(1xe2x86x924) O-glycosidic linkages in starch. Generally, xcex1-amylases (E.C. 3.2.1.1, xcex1-D-(1xe2x86x924)-glucan glucanohydrolase) are defined as endo-acting enzymes cleaving xcex1-D-(1xe2x86x924) O-glycosidic linkages within the starch molecule in a random fashion [3]. In contrast, the exo-acting amylolytic enzymes, such as xcex2-amylases (E.C. 3.2.1.2, xcex1-D-(1xe2x86x924)-glucan maltohydrolase), and some product-specific amylases cleave the starch molecule from the non-reducing end of the substrate [4]. xcex2-Amylases, xcex1-glucosidases (E.C. 3.2.1.20, xcex1-D-glucoside glucohydrolase), glucoamylase (E.C. 3.2.1.3, xcex1-D-(1xe2x86x924)-glucan glucohydrolase), and product-specific amylases can produce malto-oligosaccharides of a specific length from starch.
Several amylases producing malto-oligosaccharides of a specific DP have been identified previously including maltohexaose-producing amylases from Klebsiella pneumonia [5, 6], Bacillus subtilis [7], B. circulans G-6 [8], B. circulans F-2 [9, 10], and B. caldovelox [11, 12]. Maltopentaose-producing amylases have been detected in B. licheniformis 584 [13] and Pseudomonas spp. [14, 15]. Furthermore, maltotetraose-producing amylases have been reported from Pseudomonas stutzeri NRRL B-3389 [16, 17], Bacillus sp. MG-4 [18] and Pseudomonas sp. IMD353 [19] and maltotriose-producing amylases from Streptomyces griseus NA-468 [20] and B. subtilis [21].
EP-B1-298,645 describes a process for preparing exo-maltotetraohydrolase of Pseudomonas stutzen or P. saccharophila using genetic engineering techniques.
U.S. Pat. No. 5,204,254 describes a native and a genetically modified exo-maltopentao-hydrolase of an alkalophilic bacterium (DSM 5853).
Very few product-specific amylases active at high pH have been identified. Examples of those that have been identified include amylases from Bacillus sp. H-167 producing maltohexaose [22, 23], from a bacterial isolate (163-26, DSM 5853) producing maltopentaose [24], from Bacillus sp. IMD370 producing maltotetraose and smaller malto-oligosaccharides [25], and from Bacillus sp. GM 8901 that initially produced maltohexaose from starch which was converted to maltotetraose during extended hydrolysis periods [26].
Starch granules heated in the presence of water undergo an order-disorder phase transition called gelatinization, where liquid is taken up by the swelling granules.
Gelatinization temperatures vary for different starches and depend for the native, unmodified starches on their biological source.
Cooling converts the gelatinised phase into a viscoelastic paste or elastic gel, depending on the starch concentration. During this process, amylose and amylopectin chains reassociate to form a more ordered structure. With time, more associations are formed and they become even more ordered. It is believed that associations of amylopectin chains DP 15-20 lead to a thermoreversible, quasi-crystalline structure.
In consequence of detrimental retrogradation, the water-holding capacity of the paste or gel system is changed with important implications on the gel texture and dietary properties.
It is known that the quality of baked bread products gradually deteriorates during storage. The crumb loses softness and elasticity and becomes firm and crumbly. This so-called staling is primarily due to the detrimental retrogradation of starch, which is understood to be a transition of the starch gelatinised during baking from an amorphous state to a quasi crystalline state. The increase in crumb firmness is often used as a measure of the staling process of bread.
Upon cooling of freshly baked bread the amylose fraction, within hours, retrogrades to develop a network. This process is beneficial in that it creates a desirable crumb structure with a low degree of firmness and improved slicing properties. More gradually crystallisation of amylopectin takes place within the gelatinised starch granules during the days after baking. In this process amylopectin is believed to reinforce the amylose network in which the starch granules are embedded. This reinforcement leads to increased firmness of the bead crumb. This reinforcement is one of the main causes of bread staling.
The rate of detrimental retrogradation or crystallisation of amylopectin depends on the length of the side chains of amylopectin. In accordance with this, cereal amylopectin retrogrades at a slower rate than amylopectin from pea or potato, which has a longer average chain length than cereal amylopectin.
This is supported by observations from amylopectin gel systems that amylopectin with average chain length of DP, i.e. degree of polymerisation, xe2x89xa611 do not crystallise at all. Furthermore the presence of very short chains of DP 6-9 seems to inhibit the crystallisation of surrounding longer side chains probably because of steric hindrance. Thereby these short chains seem to have a strong anti-detrimental retrogradation effect. In accordance with this, amylopectin retrogradation is directly proportional to the mole fraction of side chains with DP 14-24 and inversely proportional to the mole fraction of side chains with DP 6-9.
In wheat and other cereals the external side chains in amylopectin are in the range of DP 12-19. Thus, enzymatic hydrolysis of the amylopectin side chains can markedly reduce their crystallisation tendencies.
It is known in the art to retard the staling of bread by using glucogenic and maltogenic exo-amylasesxe2x80x94such as amylogycosidases which hydrolyse starch by releasing glucosexe2x80x94and maltogenic exoamylases or xcex2-amylasesxe2x80x94which hydrolyse starch by releasing maltose from the non-reducing chain ends.
In this respect, Jakubczyk et al. (Zesz. Nauk. Sck. GI. Gospod Wiejsk. Warzawie, Technol. Reino-Spozyw, 1973, 223-235) reported that amyloglucosidase can retard staling of bread baked on wheat flour.
JP-62-79745 and JP-62-79746 state that the use of a xcex2-amylase produced by Bacillus stearothermophilus and Bacillus megaterium, respectively may be effective in retarding staling of starchy foods, including bread.
EP-A-412,607 discloses a process for the production of a bread product having retarded staling properties by the addition to the dough of a thermostable exoamylase, which is not inactivated before gelatinization. Only amyloglycosidases and xcex2-amylases are listed as suitable exoamylases to be used. The exoamylase is in an amount which is able to modify selectively the crystallisation properties of the amylopectin component during baking by splitting off glucose or maltose from the non-reducing ends of amylose and amylopectin. According to EP-A-412,607, the exoamylase selectively reduces the crystallisation properties of amylopectin, without substantially effecting the crystallisation properties of amylose.
EP-A-494,233 discloses the use of a maltogenic exoamylase to release maltose in the xcex1-configuration and which is not inactivated before gelatinization in a process for the production of a baked product having retarded staling properties. Only a maltogenic xcex1-amylase from Bacillus strain NCIB 11837 is specifically disclosed. Apparently, the maltogenic exoamylase hydrolyses (1xe2x86x924)-xcex1-glucosidic linkages in starch (and related polysaccharides) by removing xcex1-maltose units from the non-reducing ends of the polysaccharide chains in a stepwise manner.
Thus, the prior art teaches that certain glucogenic exoamylases and maltogenic exoamylases can provide an antistaling effect by selectively reducing the detrimental retrogradation tendencies of amylopectin through shortening of the amylopectin side chains.
Nevertheless, there is still a need to provide different and effective, preferably more effective, means for retarding the detrimental retrogradation, such as retarding the staling, of starch products, in particular baked products, more in particular bread products.
The present invention provides a process for making a starch product that has a retarded detrimental retrogradation property.
The present invention also provides enzymes that are useful in the process of the present invention.
The enzymes of the present invention are amylase enzymes. More in particular, the enzymes of the present invention are non-maltogenic exoamylase enzymes.
It is to be noted that non-maltogenic exoamylases have not hitherto been used to retard the detrimental retrogradation of starch products, let alone to retard staling in baked products.
Thus, according to a first aspect of the present invention there is provided a process for making a starch product comprising adding to a starch medium a non-maltogenic exoamylase that is capable of hydrolysing starch by cleaving off linear maltooligosaccharides, predominantly consisting of from four to eight D-glucopyranosyl units, from the non-reducing ends of the side chains of amylopectin.
Addition of the non-maltogenic exoamylase to the starch medium may occur during and/or after heating of the starch product.
Thus, according to a second aspect of the present invention there is provided a baked product obtained by the process according to the present invention.
Thus, according to a third aspect of the present invention there is provided an improver composition for a dough; wherein the composition comprises a non-maltogenic exoamylase, and at least one further dough ingredient or dough additive.
Thus, according to a fourth aspect of the present invention there is provided the use of a non-maltogenic exoamylase in a starch product to retard the detrimental retrogradation of the starch product.
Thus, according to a fifth aspect of the present invention there is provided a novel non-maltogenic exoamylase.
These and other aspects of the present invention are presented in the acompanying claims. In addition, these and other aspects of the present invention, as well as preferred aspects thereof, are presented and dicussed below.
General Definitions
Thus, the present invention relates to the use of proteins that are capable of retarding the detrimental retrogradation of starch media, in particular starch gels.
In one preferred aspect, the present invention relates to the use of proteins that are capable of retarding the staling of starch.
In another aspect, the present invention relates to the use of proteins that are capable of retarding the detrimental retrogradation of starch media, such as starch gels.
In accordance with the present invention, the term xe2x80x9cstarchxe2x80x9d means starch per se or a component thereof, especially amylopectin.
In accordance with the present invention, the term xe2x80x9cstarch mediumxe2x80x9d means any suitable medium comprising starch.
The term xe2x80x9cstarch productxe2x80x9d means any product that contains or is based on or is derived from starch.
Preferably, the starch product contains or is based on or is derived from starch obtained from wheat flour.
The term xe2x80x9cwheat flourxe2x80x9d as used herein is a synonym for the finely-ground meal of wheat or other grain. Preferably, however, the term means flour obtained from wheat per se and not from another grain. Thus, and unless otherwise expressed, references to xe2x80x9cwheat flourxe2x80x9d as used herein preferably mean references to wheat flour per se as well as to wheat flour when present in a medium, such as a dough.
A preferred flour is wheat flour or rye flour or mixtures of wheat and rye flour. However, dough comprising flour derived from other types of cereals such as for example from rice, maize, barley, and durra are also contemplated.
Preferably, the starch product is a bakery product.
More preferably, the starch product is a bread product.
Even more preferably, the starch product is a baked farinaceous bread product.
The term xe2x80x9cbaked farinaceous bread productxe2x80x9d is understood to refer to any baked product based on ground cereals and baked on a dough obtainable by mixing flour, water, and a leavening agent under dough forming conditions. It is, however, within the scope of the present invention that further components can be added to the dough mixture.
The term xe2x80x9camylasexe2x80x9d is used in its normal sensexe2x80x94e.g. an enzyme that is inter alia capable of catalysing the degradation of starch. In particular they are hydrolases which are capable of cleaving xcex1-D-(1xe2x86x924) O-glycosidic linkages in starch.
The term xe2x80x9cnon-maltogenic exoamylase enzymexe2x80x9d means the enzyme does not initially degrade starch to substantial amounts of maltose. In a highly preferred aspect, the term also means the enzyme does not initially degrade starch to substantial amounts of maltose and glucose.
Before the present invention, non-maltogenic exoamylase enzymes had not been suggested for retarding the detrimental retrogradation of starch media, in particular starch gels.
A suitable assay for determining amylase activity in accordance with the present invention is presented later. For convenience, this assay is called the xe2x80x9cAmylase Assay Protocolxe2x80x9d.
Thus, preferably, the term xe2x80x9cnon-maltogenic exoamylase enzymexe2x80x9d means that the enzyme does not initially degrade starch to substantial amounts of maltose as analysed in accordance with the product determination procedure as described in the xe2x80x9cAmylase Assay Protocolxe2x80x9d presented herein.
In a preferred aspect, the non-maltogenic exoamylase can be characterised in that if an amount of 0.7 units of said non-maltogenic exoamylase were to incubated for 15 minutes at a temperature of 50xc2x0 C. at pH 6.0 in 4 ml of an aqueous solution of 10 mg preboiled waxy maize starch per ml buffered solution containing 50 mM 2-(N-morpholino)ethane sulfonic acid and 2 mM calcium chloride then the enzyme would yield hydrolysis product(s) that would consist of one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the said hydrolysis products would consist of linear maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D-glucopyranosyl units.
For ease of reference, and for the present purposes, the feature of incubating an amount of 0.7 units of the non-maltogenic exoamylase for 15 minutes at a temperature of 50xc2x0 C. at pH 6.0 in 4 ml of an aqueous solution of 10 mg preboiled waxy maize starch per ml buffered solution containing 50 mM 2-(N-morpholino)ethane sulfonic acid and 2 mM calcium chloride, may be referred to as the xe2x80x9cwaxy maize starch incubation testxe2x80x9d.
Thus, alternatively expressed, a preferred non-maltogenic exoamylase is characterised as having the ability in the waxy maize starch incubation test to yield hydrolysis product(s) that would consist of one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the said hydrolysis product(s) would consist of linear maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D-glucopyranosyl units.
The hydrolysis products in the waxy maize starch incubation test include one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose. The hydrolysis products in the waxy maize starch incubation test may also include other hydrolytic products. Nevertheless, the % weight amounts of linear maltooligosaccharides of from three to ten D-glucopyranosyl units are based on the amount of the hydrolysis product that consists of one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose. In other words, the % weight amounts of linear maltooligosaccharides of from three to ten D-glucopyranosyl units are not based on the amount of hydrolysis products other than one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and glucose.
The hydrolysis products can be analysed by any suitable means. For example, the hydrolysis products may be analysed by anion exchange HPLC using a Dionex PA 100 column with pulsed amperometric detection and with, for example, known linear maltooligosaccharides of from glucose to maltoheptaose as standards.
For ease of reference, and for the present purposes, the feature of analysing the hydrolysis product(s) using anion exchange HPLC using a Dionex PA 100 column with pulsed amperometric detection and with known linear maltooligosaccharides of from glucose to maltoheptaose used as standards, can be referred to as xe2x80x9canalysing by anion exchangexe2x80x9d. Of course, and as just indicated, other analytical techniques would suffice, as well as other specific anion exchange techniques.
Thus, alternatively expressed, a preferred non-maltogenic exoamylase is characterised as having the ability in a waxy maize starch incubation test to yield hydrolysis product(s) that would consist of one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose, said hydrolysis products being capable of being analysed by anion exchange; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the said hydrolysis product(s) would consist of linear maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D-glucopyranosyl units.
As used herein with respect to the present invention, the term xe2x80x9clinear malto-oligosaccharidexe2x80x9d is used in the normal sense as meaning 2-10 units of xcex1-D-glucopyranose linked by an xcex1-(1xe2x86x924) bond.
The term xe2x80x9cobtainable from P. saccharophilaxe2x80x9d means that the enzyme need not necessarily be obtained from P. saccharophila. Instead, the enzyme could be prepared by use of recombinant DNA techniques.
The term xe2x80x9cfunctional equivalent thereofxe2x80x9d in relation to the enzyme being obtainable from P. saccharophila means that the functional equivalent could be obtained from other sources. The functionally equivalent enzyme may have a different amino acid sequence but will have non-maltogenic exoamylase activity. The functionally equivalent enzyme may have a different chemical structure and/or formula but will have non-maltogenic exoamylase activity. The functionally equivalent enzyme need not necessarily have exactly the same non-maltogenic exoamylase activity as the non-maltogenic exoamylase enzyme obtained from P. saccharophila. For some applications, preferably, the functionally equivalent enzyme has at least the same activity profile as the enzyme obtained from P. saccharophila. 
The term xe2x80x9cobtainable from Bacillus clausiixe2x80x9d means that the enzyme need not necessarily be obtained from Bacillus clausii. Instead, the enzyme could be prepared by use of recombinant DNA techniques.
The term xe2x80x9cfunctional equivalent thereofxe2x80x9d in relation to the enzyme being obtainable from Bacillus clausii means that the functional equivalent could be obtained from other sources. The functionally equivalent enzyme may have a different amino acid sequence but will have non-maltogenic exoamylase activity. The functionally equivalent enzyme may have a different chemical structure and/or formula but will have non-maltogenic exoamylase activity. The functionally equivalent enzyme need not necessarily have exactly the same non-maltogenic exoamylase activity as the non-maltogenic exoamylase enzyme obtained from Bacillus clausii. For some applications, preferably, the functionally equivalent enzyme has at least the same activity profile as the enzyme obtained from Bacillus clausii (such as the reactivity profile shown in FIG. 7).
General Comments
The present invention is based on the surprising finding that non-maltogenic exoamylases are highly effective in retarding or reducing detrimental retrogradation, such as staling, in starch products, in particular baked products.
We have found that non-maltogenic exoamylases according to the present invention can be more effective in retarding detrimental retrogradation, such as staling, in bread than the glucogenic and maltogenic exoamylases.
The reduction of detrimental retrogradation can be measured by standard techniques known in the art. By way of example, some techniques are presented later on in the section titled xe2x80x9cAssay for the Measurement of Retrogradationxe2x80x9d.
In our studies, we have found that by incorporating a sufficient amount of activity of a non-maltogenic exoamylase, like for instance a exo-maltotetraohydrolase (EC 3.2.1.60), which has a sufficient thermostability, into a dough there is provided baked products with reduced, in some cases significantly reduced, detrimental retrogradation compared to that of a control bread, such as under storage conditions. In contrast, the reducing effect on detrimental retrogradation of incorporating the same amount of activity of a maltogenic exoamylase with a comparable thermostability to that of the non-maltogenic exoamylase is significantly less. Thus, the anti-retrogradation effect of non-maltogenic exoamylase is more efficient than that of a maltogenic exoamylase. We believe that this difference may be, in part, due to the extent to which the amylopectin side chains are shortened. We also believe that the anti-retrogradation effect may be even more pronounced when using a non-maltogenic exoamylase according to the invention which releases maltoheptaose and/or maltooctaose and/or maltohexose.
In our studies we have also purified and characterised a product-specific amylase active at high pH producing maltohexaose. This amylase was isolated from an alkali-tolerant strain of Bacillus clausii BT-21.
Furthermore, we have found that the retardation of detrimental retrogradation that is obtainable by using non-maltogenic exoamylases according to the present invention is dose responsive over a very wide range. This is in contrast to the effect from maltogenic exoamylases, which is rather limited and has a strongly decreasing dose response.
Amylases
In one aspect, the present invention provides the use of certain amylases to prepare starch products, such as bakery products. In this respect, the amylasesxe2x80x94which are non-maltogenic exoamylasesxe2x80x94retard or reduce the staling properties (i.e. lower the rate of staling) of the starch product, in particular a baked farinaceous bread product.
Preferably, the amylase is in an isolated form and/or in a substantially pure form. Here, the term xe2x80x9cisolatedxe2x80x9d means that the enzyme is not in its natural environment.
As indicated above, the non-maltogenic exoamylase enzyme of the present invention does not initially degrade starch to substantial amounts of maltose.
According to the present invention, the non-maltogenic exoamylase is capable of cleaving off linear maltooligosaccharides, predominantly consisting of from four to eight D-glucopyranosyl units, from the non-reducing ends of the side chains of amylopectin. Non-maltogenic exoamylases having this characteristic and which are suitable for use in the present invention are identified by their ability to hydrolyse gelatinised waxy maize starch in the model system presented in the Amylase Assay Protocol (infra).
When incubated 15 min. under the described conditions in the Amylase Assay Protocol, the non-maltogenic exoamylases which are suitable for use according to the present invention would yield a hydrolysis product(s) that would consist of one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose, such that the product pattern of that hydrolysis product would consist of at least 60%, in particular at least 70%, more preferably at least 80% and most preferably at least 90% by weight of starch hydrolysis degradation products other than maltose and glucose.
For a preferred aspect of the present invention, the non-maltogenic exoamylases which are suitable for use according to the present invention would provide when incubated 15 min. under the described conditions for the waxy maize starch incubation test the said hydrolysis product, such that the hydrolysis product would have a product pattern of at least 60%, in particular at least 70%, more preferably at least 80% and most preferably at least 90% by weight of linear malto-oligosaccharides of from three to ten D-glucopyranosyl units, in particular linear maltooligosaccharides consisting of from four to eight D-glucopyranosyl units.
In a more preferred aspect of the present invention, the said hydrolysis product in said test would have a product pattern of at least 60%, in particular at least 70%, more preferably at least 80% and most preferably at least 85% by weight of linear maltooligosaccharides of 4 or 6 D-glucopyranosyl units.
In a more preferred aspect of the present invention, the said hydrolysis product in said test would have a product pattern of at least 60%, in particular at least 70%, more preferably at least 80% and most preferably at least 85% by weight linear maltooligosaccharides of 4 D-glucopyranosyl units.
In a more preferred aspect of the present invention, the said hydrolysis product in said test would have a product pattern of at least 60%, in particular at least 70%, more preferably at least 80% and most preferably at least 85% by weight of linear maltooligosaccharides of 6 D-glucopyranosyl units.
Preferentially, the non-maltogenic exoamylase does not substantially hydrolyze its primary products to convert them to glucose, maltose and maltotriose. If that were the case, the primary products would compete as substrates with the amylopectin non-reducing chain ends for the enzyme, so that its anti-retrogradation efficiency would be reduced.
Thus, preferentially, the non-maltogenic exoamylase when incubated for 300 min. under conditions similar to the waxy maize starch incubation test but wherein the 15 min. period is extended to 300 min.xe2x80x94as an aside, and for convenience for the present purposes, this modified waxy maize starch incubation test may be called the xe2x80x9cextended waxy maize starch incubation testxe2x80x9dxe2x80x94would still yield the said hydrolyis product wherein the hydrolysis product would have a product pattern of at least 50%, in particular at least 60%, more preferably at least 70% and most preferably at least 80% by weight of from four to eight D-glucopyranosyl units.
By way of example, a non-maltogenic exoamylase useful in the process of the present invention can be characterised in that it has the ability in a waxy maize starch incubation test to yield hydrolysis product(s) that would consist of one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the said hydrolysis product(s) would consist of linear maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D-glucopyranosyl units; and wherein the enzyme is obtainable from P. saccharophila or is a functional equivalent thereof.
By way of further example, another non-maltogenic exoamylase useful in the process of the present invention can be characterised in that it has the ability in a waxy maize starch incubation test to yield hydrolysis product(s) that would consist of one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the said hydrolysis product(s) would consist of linear maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D-glucopyranosyl units; wherein the enzyme is obtainable from Bacillus clausii or is a functional equivalent thereof; and wherein the enzyme has a molecular weight of about 101,000 Da (as estimated by sodium dodecyl sulphate polyacrylamide electrophoresis) and/or the enzyme has an optimum of activity at pH 9.5 and 55xc2x0 C.
Preferably, the non-maltogenic exoamylases which are suitable for use according to the present invention are active during baking and hydrolyse starch during and after the gelatinization of the starch granules which starts at temperatures of about 55xc2x0 C. The more thermostable the non-maltogenic exoamylase is the longer time it can be active and thus the more antistaling effect it will provide. However, during baking above temperatures of about 85xc2x0 C. the non-maltogenic exoamylase is preferentially gradually inactivated so that there is substantially no activity after the baking process in the final bread. Therefore preferentially the non-maltogenic exoamylases suitable for use according to the present invention have an optimum temperature above 45xc2x0 C. and below 98xc2x0 C. when incubated for 15 min. at 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90xc2x0 C. in a test tube with 4 ml of 10 mg/ml waxy maize starch in 50 mM MES, 2 mM calcium chloride, pH 6.0 prepared as described above and assayed for release of hydrolysis products as described above. Preferably the optimum temperature of the non-maltogenic exoamylase is above 55xc2x0 C. and below 95xc2x0 C. and even more preferably it is above 60xc2x0 C. and below 90xc2x0 C.
Non-maltogenic exoamylases which may be found to be less thermostable can be improved by using protein engineering to become more thermostable and thus better suited for use according to present the invention. Thus the use of non-maltogenic exoamylases modified to become more thermostable by protein engineering is encompassed by the present invention.
It is known that some non-maltogenic exoamylases can have some degree of endoamylase activity. In some cases, this type of activity may need to be reduced or eliminated since endoamylase activity can possibly negatively effect the quality of the final bread product by producing a sticky or gummy crumb due to the accumulation of branched dextrins.
Thus, in a preferred aspect, the non-maltogenic exoamylase of the present invention will have less than 0.5 endoamylase units (EAU) per unit of exoamylase activity.
Preferably the non-maltogenic exoamylases which are suitable for use according to the present invention have less than 0.05 EAU per unit of exoamylase activity and more preferably less than 0.01 EAU per unit of exoamylase activity.
The endoamylase units can be determined by use of the Endoamylase Assay Protocol presented below.
Examples of non-maltogenic exoamylases suitable for use according to the present invention include exo-maltotetraohydrolase (E.C.3.2.1.60), exo-maltopentaohydrolase and exo-maltohexaohydrolase (E.C.3.2.1.98) which hydrolyze 1,4xcex1-glucosidic linkages in amylaceous polysaccharides so as to remove successive residues of maltotetraose, maltopentaose or maltohexaose, respectively, from the non-reducing chain ends. Examples are exo-maltotetrao-hydrolases of Pseudomonas saccharophila and P. stutzeri (EP-0 298 645 B1), exo-maltopentaohydrolases of an alkaliphilic Gram-positive bacterium (U.S. Pat. No. 5,204,254) and of Pseudomonas sp. (Shida et al., Biosci. Biotechnol. Biochem., 1992, 56, 76-80) and exo-maltohexaohydrolases of Bacillus sp. #707 (Tsukamoto et al., Biochem. Biophys. Res. Commun., 1988, 151, 25-31), B. circulans F2 (Taniguchi, ACS Symp., 1991, Ser. 458, 111-124) and Aerobacter aerogenes (Kainuma et al., Biochim. Biophys. Acta, 1975, 410, 333-346).
Another example of a non-maltogenic exoamylase suitable for use according to the invention is the exoamylase from an alkalophilic Bacillus strain, GM8901 (28). This is a non-maltogenic exoamylase which produces maltotetraose as well as maltopentaose and maltohexaose from starch.
Furthermore, non-maltogenic exoamylases suitable for use according to the present invention also include exo-maltoheptaohydrolase or exo-maltooctaohydrolase which hydrolyze 1,4-xcex1-glucosidic linkages in amylaceous polysaccharides so as to remove residues of maltoheptaose or maltooctaose, respectively, from the non-reducing chain ends. Exo-maltoheptaohydrolase and exo-maltooctaohydrolase can be found either by screening wild type strains or can be developed from other amylolytic enzymes by protein engineering. Thus, non-maltogenic exoamylases developed by protein engineering from other amylolytic enzymes to become non-maltogenic exoamylases are also suitable for use in the present invention.
Novel Amylase
In one aspect, the present invention also provides a novel amylase that is suitable for preparing starch products according to the present invention, such as bakery products. The novel amylase of the present invention is a non-maltogenic exoamylase. In our studies, we have chararacterised this new amylase that is suitable for the preparation of foodstuffs, in particular doughs for use in the preparation of bakery products.
Thus, the present invention also provides a non-maltogenic exoamylase, wherein the non-maltogenic exoamylase is further characterised in that it has the ability in a waxy maize starch incubation test to yield hydrolysis product(s) that would consist of one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the said hydrolysis product(s) would consist of linear maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D-glucopyranosyl units; wherein the enzyme is obtainable from Bacillus clausii or is a functional equivalent thereof; and wherein the enzyme has a molecular weight of about 101,000 Da (as estimated by sodium dodecyl sulphate polyacrylamide electrophoresis) and/or the enzyme has an optimum of activity at pH 9.5 and 55xc2x0 C.
Preferably, the amylase is in an isolated form and/or in a substantially pure form. Here, the term xe2x80x9cisolatedxe2x80x9d means that the enzyme is not in its natural environment.
Antibodies
The enzymes of present invention can also be used to generate antibodiesxe2x80x94such as by use of standard techniques. Thus, antibodies to each enzyme according to the present invention may be raised. The or each antibody can be used to screen for other suitable amylase enzymes according to the present invention. In addition, the or each antibody may be used to isolate amounts of the enzyme of the present invention.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, etc. may be immunized by injection with the inhibitor or any portion, variant, homologue, fragment or derivative thereof or oligopeptide which retains immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund""s, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (Bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants which may be employed.
Monoclonal antibodies to the enzyme may be even prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, pp 77-96). In addition, techniques developed for the production of xe2x80x9cchimeric antibodiesxe2x80x9d, the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison et al (1984) Proc Natl Acad Sci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda et al (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,779) can be adapted to produce inhibitor specific single chain antibodies.
Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991; Nature 349:293-299).
Improver Composition
As indicated, one aspect of the present invention relates to an improver composition for a starch product, in particular a dough and/or a baked farinaceous bread product made from the dough.
The improver composition comprises a non-maltogenic exoamylase according to the present invention and at least one further dough ingredient or dough additive.
According to the present invention the further dough ingredient or dough additive can be any of the dough ingredients and dough additives which are described above.
Expediently, the improver composition is a dry pulverulent composition comprising the non-maltogenic exoamylase according to the invention admixed with at least one further ingredient or additive. However, the improver composition may also be a liquid preparation comprising the non-maltogenic exoamylase according to the invention and at least one further ingredient or additive dissolved or dispersed in water or other liquid. It will be understood that the amount of enzyme activity in the improver composition will depend on the amounts and types of the further ingredients and additives which form part of the improver composition.
Optionally, the improver composition may be in the form of a complete mixture, a so-called pre-mixture, containing all of the dry ingredients and additives for making a particular baked product.
Preparation of Starch Products
In accordance with one aspect of the present invention, the process comprises forming the starch product by adding a suitable non-maltogenic exoamylase enzyme, such as one of the novel non-maltogenic exoamylase enzymes presented herein, to a starch medium.
If the starch medium is a dough, then the dough is prepared by mixing together flour, water, the non-maltogenic exoamylase according to the invention and other possible ingredients and additives.
By way of further example, if the starch product is a baked farinaceous bread product (which is a highly preferred embodiment), then the process comprises mixingxe2x80x94in any suitable orderxe2x80x94flour, water, and a leavening agent under dough forming conditions and further adding a suitable non-maltogenic exoamylase enzyme.
The leavening agent may be a chemical leavening agent such as sodium bicarbonate or any strain of Saccharomyces cerevisiae (Baker""s Yeast).
The non-maltogenic exoamylase can be added together with any dough ingredient including the water or dough ingredient mixture or with any additive or additive mixture.
The dough can be prepared by any conventional dough preparation method common in the baking industry or in any other industry making flour dough based products.
Baking of farinaceous bread products such as for example white bread, bread made from bolted rye flour and wheat flour, rolls and the like is typically accomplished by baking the bread dough at oven temperatures in the range of from 180 to 250xc2x0 C. for about 15 to 60 minutes. During the baking process a steep temperature gradient (200xe2x86x92120xc2x0 C.) is prevailing in the outer dough layers where the characteristic crust of the baked product is developed. However, owing to heat consumption due to steam generation, the temperature in the crumb is only close to 100xc2x0 C. at the end of the baking process.
The non-maltogenic exoamylase can be added as a liquid preparation or as a dry pulverulent composition either comprising the enzyme as the sole active component or in admixture with one or more additional dough ingredient or dough additive.
In order to improve further the properties of the baked product and impart distinctive qualities to the baked product further dough ingredients and/or dough additives may be incorporated into the dough. Typically, such further added components may include dough ingredients such as salt, grains, fats and oils, sugar, dietary fibre substances, milk powder, gluten and dough additives such as emulsifiers, other enzymes, hydrocolloids, flavouring agents, oxidising agents, minerals and vitamins.
The emulsifiers are useful as dough strengtheners and crumb softeners. As dough strengtheners, the emulsifiers can provide tolerance with regard to resting time and tolerance to shock during the proofing. Furthermore, dough strengtheners will improve the tolerance of a given dough to variations in the fermentation time. Most dough strengtheners also improve on the oven spring which means the increase in volume from the proofed to the baked goods. Lastly, dough strengtheners will emulsify any fats present in the recipe mixture.
The crumb softening, which is mainly a characteristic of the monoglycerides, is attributed to an interaction between the emulsifier and the amylose fraction of the starch leading to formation of insoluble inclusion complexes with the amylose which will not recrystallize upon cooling and which will not therefore contribute to firmness of the bread crumb.
Suitable emulsifiers which may be used as further dough additives include lecithin, polyoxyethylene stearat, mono- and diglycerides of edible fatty acids, acetic acid esters of mono- and diglycerides of edible fatty acids, lactic acid esters of mono- and diglycerides of edible fatty acids, citric acid esters of mono- and diglycerides of edible fatty acids, diacetyl tartaric acid esters of mono- and diglycerides of edible fatty acids, sucrose esters of edible fatty acids, sodium stearoyl-2-lactylate, and calcium stearoyl-2-lactylate.
Other enzymes which are useful as further dough additives include as examples oxidoreductases, such as glucose oxidase, hexose oxidase, and ascorbate oxidase, hydrolases, such as lipases and esterases as well as glycosidases like xcex1- amylase, pullulanase, and xylanase. Oxidoreductases, such as for example glucose oxidase and hexose oxidase, can be used for dough strengthening and control of volume of the baked products and xylanases and other hemicellulases may be added to improve dough handling properties, crumb softness and bread volume. Lipases are useful as dough strengtheners and crumb softeners and xcex1-amylases and other amylolytic enzymes may be incorporated into the dough to control bread volume and further reduce crumb firmness.
The amount of the non-maltogenic exoamylase according to the present invention that is added is normally in an amount which results in the presence in the finished dough of 50 to 100,000 units per kg of flour, preferably 100 to 50,000 units per kg of flour. In useful embodiments of the present invention, the amount is in the range of 200 to 20,000 units per kg of flour.
In the present context, 1 unit of the non-maltogenic exoamylase is defined as the amount of enzyme which releases hydrolysis products equivalent to 1 xcexcmol of reducing sugar per min. when incubated at 50xc2x0 C. in a test tube with 4 ml of 10 mg/ml waxy maize starch in 50 mM MES, 2 mM calcium chloride, pH 6.0 as described hereinafter.
Foodstuffs Prepared with Amylases
The present invention provides suitable amylases for use in the manufacture of a foodstuff. Typical foodstuffs, which also include animal feed, include dairy products, meat products, poultry products, fish products and bakery products.
Preferably, the foodstuff is a bakery product, such as the bakery products described above. Typical bakery (baked) products incorporated within the scope of the present invention include breadxe2x80x94such as loaves, rolls, buns, pizza bases etc.xe2x80x94pretzels, tortillas, cakes, cookies, biscuits, krackers etc.
Amylase Assay Protocol
The following system is used to characterize non-maltogenic exoamylases which are suitable for use according to the present invention.
By way of initial background information, waxy maize amylopectin (obtainable as WAXILYS 200 from Roquette, France) is a starch with a very high amylopectin content (above 90%).
20 mg/ml of waxy maize starch is boiled for 3 min. in a buffer of 50 mM MES (2-(N-morpholino)ethanesulfonic acid), 2 mM calcium chloride, pH 6.0 and subsequently incubated at 50xc2x0 C. and used within half an hour.
One unit of the non-maltogenic exoamylase is defined as the amount of enzyme which releases hydrolysis products equivalent to 1 xcexcmol of reducing sugar per min. when incubated at 50xc2x0 C. in a test tube with 4 ml of 10 mg/ml waxy maize starch in 50 mM MES, 2 mM calcium chloride, pH 6.0 prepared as described above.
Reducing sugars are measured using maltose as standard and using the dinitrosalicylic acid method of Bernfeld, Methods Enzymol., (1954), 1, 149-158 or another method known in the art for quantifying reducing sugars.
The hydrolysis product pattern of the non-maltogenic exoamylase is determined by incubating 0.7 units of non-maltogenic exoamylase for in a test tube with 4 ml of 10 mg/ml waxy maize starch in the buffer prepared as described above. The reaction is stopped by immersing the test tube for 3 min. in a boiling water bath.
The hydrolysis products are analyzed and quantified by anion exchange HPLC using a Dionex PA 100 column with sodium acetate, sodium hydroxide and water as eluents, with pulsed amperometric detection and with known linear maltooligo-saccharides of from glucose to maltoheptaose as standards. The response factor used for maltooctaose to maltodecaose is the response factor found for maltoheptaose.
Endoamylase Assay Protocol
0.75 ml of enzyme solution is incubated with 6.75 ml of 0.5% (w/v) of AZCL-amylose (azurine cross-linked amylose available from Megazyme, Ireland) in 50 mM MES (2-(N-morpholino)ethanesulfonic acid), 2 mM calcium chloride, pH 6.0 at 50xc2x0 C. After 5, 10, 15, 20 and 25 minutes, respectively 1.0 ml of reaction mix is transferred to 4.0 ml of stop solution consisting of 4% (w/v) TRIS (Tris(hydroxy-methyl)aminomethane).
The stopped sample is filtered through a Whatman No. 1 filter and its optical density at 590 nm is measured against distilled water. The enzyme solution assayed should be diluted so that the optical density obtained is a linear function of time. The slope of the line for optical density versus time is used to calculate the endoamylase activity relative to the standard GRINDAMYL(trademark) A1000 (available from Danisco Ingredients), which is defined to have 1000 endoamylase units (EAU) per g.
Assays for Measurement of Retrogradation (inc. Staling)
For evaluation of the antistaling effect of the non-maltogenic exoamylase of the present invention, the crumb firmness can be measured 1, 3 and 7 days after baking by means of an Instron 4301 Universal Food Texture Analyzer or similar equipment known in the art.
Another method used traditionally in the art and which is used to evaluate the effect on starch retrogradation of a non-maltogenic exoamylase according to the present invention is based on DSC (differential scanning calorimetry). Hereby the melting enthalpy of retrograded amylopectin in bread crumb or crumb from a model system dough baked with or without enzymes (control) is measured. The DSC equipment applied in the described examples is a Mettler-Toledo DSC 820 run with a temperature gradient of 10xc2x0 C. per min. from 20 to 95xc2x0 C. For preparation of the samples 10-20 mg of crumb are weighed and transferred into Mettler-Toledo aluminium pans which then are hermetically sealed.
The model system doughs used in the described examples contain standard wheat flour and optimal amounts of water or buffer with or without the non-maltogenic exoamylase according to the present invention. They are mixed in a 10 or 50 g Brabender Farinograph for 6 or 7 min., respectively. Samples of the doughs are placed in glass test tubes (15*0.8 cm) with a lid. These test tubes are subjected to a baking process in a water bath starting with 30 min. incubation at 33xc2x0 C. followed by heating from 33 to 95xc2x0 C. with a gradient of 1.1xc2x0 C. per min. and finally a 5 min. incubation at 95xc2x0 C. Subsequently, the tubes are stored in a thermostat at 20xc2x0 C. prior to DSC analysis.
Summary
In summary the present invention is based on the surprising finding that non-maltogenic exoamylasesxe2x80x94which hydrolyse starch by cleaving off linear maltooligosaccharides in the range of four to eight D-glucopyranosyl units from the non-reducing chain ends of amylopectin and which preferably have a sufficient degree of thermostabilityxe2x80x94are highly effective in retarding or reducing detrimental retrogradation in baked products.
Deposits
The following sample was deposited in accordance with the Budapest Treaty at the recognised depositary DSMZ (Deutsche Sammiung von Mikrooganismen und Zellkulturen GmbH of Mascheroder Weg 1b, D-38124 Braunschweig) on Mar. 12 1999:
BT-21 DSM number DSM 12731
The present invention also encompasses sequences derivable and/or expressable from those deposits and embodiments comprising the same, as well as active fragments thereof.